Two-color STED Sample Preparation

Staining:

Channel

Best

Better

Good

Red

STAR 580

Atto 590

Atto 594, Dylight 594

Dark Red

STAR 635 & STAR 635P

Atto 647N

Secondary antibodies conjugated to appropriate fluorophores for both channels are available from Abberior, Enzo Life Sciences, Rockland Inc, Active Motif, Hypermol, Sigma-Aldrich and Axxora. Download the file to see which companies have what conjugates available.

Prolong Gold (WITHOUT DAPI) with 2.5% DABCO is the best option for the mounting medium for samples where you want to image within 10 microns of the coverslip. Note that Prolong Gold takes approximately 24 hours at room temperature in the dark to cure. If attempting to image deeper than 10 microns, Mowiol with 2.5% DABCO is recommended. Note that if you want to just image at one depth you may not need to add DABCO, but it may be necessary if you want to do z-stacks of your samples.

Tissue sections can be imaged, but the resolution decays with tissue depth and one must be careful about the axial resolution (see 6). If possible it is best to cut very thin tissue sections (<10 microns thick, depending on the type of tissue, i.e. cut highly scattering tissue thinner) with a cryo-ultramicrotome from thicker sections. See this paper.

Coverslips of thickness #1.5 (Note: it is very important to use the proper thickness coverslip or you will have reduced resolution) should be mounted in the center of the slide, with the length of the coverslip being 40 mm or less. The width of the coverslip doesn’t matter as long as it fits on the slide.

The STED microscope currently doesn’t have resolution enhancement in z (axial), so z-resolution is the same as confocal, about 600 nm in this case. This will cause serious problems if you have more than one of the bio-structures of interest in that 600 nm thickness as you will not be able to resolve them, even if they are far enough apart in the lateral direction to resolve. Thus you must either very thinly slice your samples, have inherently thin samples (i.e. tubulin or actin in very flat parts of cells) or have very sparse labeling or we will not get super-resolution images.

The increased resolution of STED microscopy, like any sub-diffraction microscopy, may disclose shortcomings of the sample preparation that are concealed by the lower resolution of conventional (confocal) microscopy. Samples must be well labeled, very bright and non-bleaching in confocal mode in order to provide good STED images.